Abstracts

We reported that the mutation of the GABA[sub]A [/sub]Receptor (GABAR) [alpha]1 subunit ([alpha]1(A322D)) linked to autosomal dominant juvenile myoclonic epilepsy substantially reduces [alpha]1(A322D) protein expression. We reported recently that this mutation causes [alpha]1(A322D) to be retained in the endoplasmic reticulum (ER) and subjected to ER associated degradation (ERAD), a process that retrotranslocates proteins from the ER to the cytosol for degradation by the proteosome. Here we determined if [alpha]1(A322D) expression can be altered by proteosomal inhibition. The construction of green and yellow fluorescent protein (FP) tagged [alpha]1-FP and [alpha]1(A322D)-FP subunits was described previously. HEK293T cells were transfected with [beta]2 and [gamma]2 subunits and either wild type, heterozygous, or homozygous [alpha]1-FP subunits. Twenty-four hours after transfection, the cells were incubated in the presence or absence of 20 [mu]M lactacystin (LAC) for an additional twelve hours. GFP fluorescence from whole cell lysates was measured in a fluorescence spectrometer. Cell lysates were also analyzed via Western blot and the relative proportions of glycosylated and unglycosylated [alpha]1-YFP and [alpha]1(A322D)-YFP subunits were quantified as described previously. Live cells, treated or untreated with LAC, were incubated with the membrane marker, FM 4-64, and imaged with confocal microscopy and the total and membrane-associated [alpha]1(A322D)-YFP was quantified. Fluorescence spectroscopy demonstrated that LAC increased the fluorescence of wild type receptors by 140 [plusmn] 12% (P = 0.03), heterozygous receptors by 150 [plusmn] 39% (P = 0.01) and homozygous receptors by 704 [plusmn] 20% (P [lt] 0.001). Western blots showed that for homozygous mutant [alpha]1(A322D)-YFP[beta]2[gamma]2 receptors, LAC was effective in increasing both the immature, unglycosylated [alpha]1(A322D)-YFP subunit (299 [plusmn] 49%) as well as the mature, glycosylated [alpha]1(A322D)-YFP subunit (202 [plusmn] 133%). In contrast, for wild type [alpha]1-YFP[beta]2[gamma]2 receptors, LAC increased expression of the immature unglycosylated [alpha]1-YFP subunit (156 [plusmn] 55%), but had little effect on the expression of the mature glycosylated [alpha]1-YFP subunit (18 [plusmn] 20%, P = 0.039). Confocal microscopic imaging of homozygous [alpha]1(A322D)-YFP[beta]2[gamma]2 receptors revealed that LAC-treated cells contained [alpha]1(A322D)-YFP in discrete intracellular inclusions, but quantification of the [alpha]1(A322D)-YFP membrane fluorescence showed that both LAC treated and untreated cells had similar fractions of total [alpha]1(A322D)-YFP on the membrane (P = 0.863). LAC increases expression of both total and membrane-associated mutant [alpha]1(A322D)-FP subunits. In contrast, LAC increases expression of only immature, nonglycosylated wild type [alpha]1-FP, thus suggesting that there is a maximal limit on the amount of [alpha]1-FP trafficked to the cell surface. Proteosomal inhibition by small molecules may represent a therapeutic target for the treatment of certain epileptic syndromes. (Supported by K08 NS44257-01, NS33300 and NS 39479.)